Metabolically engineered cells for the production of resveratrol or an oligomeric or glycosidically-bound derivative thereof

ABSTRACT

A recombinant micro-organism producing resveratrol by a pathway in which phenylalanine ammonia lyase (PAL) produces trans-cinnamic acid from phenylalanine, cinnamate 4-hydroxylase (C4H) produces 4-coumaric acid from said trans-cinnamic acid, 4-coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4-coumaroyl CoA, or in which L-phenylalanine- or tyrosine-ammonia lyase (PAL/TAL) produces 4-coumaric acid, 4-coumarate-CoA ligase (4CL) produces 4-coumaroyl CoA from said 4-coumaric acid, and resveratrol synthase (VST) produces said resveratrol from said 4-coumaroyl CoA. The micro-organism may be a yeast, fungus or bacterium including  Saccharomyces cerevisiae, E. coli, Lactococcus lactis, Aspergillus niger , or  Aspergillus oryzae.

FIELD OF THE INVENTION

This invention relates generally to the production of the polyphenolresveratrol or an oligomeric or glycosidically bound derivative thereofsuch as its β-glucoside piceid using microbial cells. Furthermore, itrelates to the use of naturally occurring or recombinant micro-organismsthat produce resveratrol or such a derivative for production of food,feed and beverages.

BACKGROUND OF THE INVENTION

Production of chemicals from micro-organisms has been an importantapplication of biotechnology. Typically, the steps in developing such abio-production method may include 1) selection of a propermicro-organism host, 2) elimination of metabolic pathways leading toby-products, 3) deregulation of desired pathways at both enzyme activitylevel and the transcriptional level, and 4) overexpression ofappropriate enzymes in the desired pathways. In preferred aspect, thepresent invention has employed combinations of the steps above toredirect carbon flow from phenylalanine or tyrosine through enzymes ofthe plant phenylpropanoid pathway which supplies the necessary precursorfor the desired biosynthesis of resveratrol.

Resveratrol (or 3,4,5-trihydroxystilbene) is a phytophenol belonging tothe group of stilbene phytoalexins, which are low-molecular-masssecondary metabolites that constitute the active defense mechanism inplants in response to infections or other stress-related events.Stilbene phytoalexins contain the stilbene skeleton(trans-1,2-diphenylethylene) as their common basic structure: that maybe supplemented by addition of other groups as well (Hart and Shrimpton,1979, Hart, 1981). Stilbenes have been found in certain trees(angio-sperms, gymnosperms), but also in some herbaceous plants (inspecies of the Myrtaceae, Vitaceae and Leguminosae families). Saidcompounds are toxic to pests, especially to fungi, bacteria and insects.Only few plants have the ability to synthesize stilbenes, or to producethem in an amount that provides them sufficient resistance to pests.

The synthesis of the basic stilbene skeleton is pursued by stilbenesynthases. So far, two enzymes have been designated as a stilbenesynthase; pinosylvine synthase and resveratrol synthase. To date, thegroundnut (Arachis hypogaea) resveratrol synthase has been characterisedin most detail, such that most of the properties are known (Schoppnerand Kindl, 1984). Substrates that are used by stilbene synthases aremalonyl-CoA, cinnamoyl-CoA or coumaroyl-CoA. These substances occur inevery plant because they are used in the biosynthesis of other importantplant constituents as well such as flavonoids, flower pigments andlipids.

Resveratrol (FIG. 1 trans-form) consists of two closely connected phenolrings and belongs therefore to the polyphenols. While present in otherplants, such as eucalyptus, spruce, and lily, and in other foods such asmulberries and peanuts, resveratrol's most abundant natural sources areVitis vinifera, -labrusca, and -muscadine (rotundifolia) grapes, whichare used to make wines. The compound occurs in the vines, roots, seeds,and stalks, but its highest concentration is in the skin (Celotti etal., 1996), which contains 50-100 μg/g. (Jang et al. 1997). During redwine vinification the grape skins are included in the must, in contrastto white wine vinification, and therefore resveratrol is found in smallquantities in red wine only. Resveratrol has, besides its antifungalproperties, been recognized for its cardioprotective- and cancerchemopreventive activities; it acts as a phytoestrogen, an inhibitor ofplatelet aggregation (Kopp et al, 1998; Gehm et al 1997; Lobo et al1995), and an antioxidant (Jang et al., 1997; Huang 1997). Theseproperties explain the so-called French Paradox, i.e. the wine-drinkingFrench have a low incidence of coronary heart disease despite alow-exercise, high-fat diet. Recently it has been shown that resveratrolcan also activate the SIR2 gene in yeast and the analogous human geneSIRT1, which both play a key role in extending life span. Ever since,attention is very much focused on the life-span extending properties ofresveratrol (Hall, 2003, Couzin, 2004).

American health associations, such as the Life Extension Foundation, arepromoting the vast beneficial effects of this drug, and therebypropelling the ideal conditions for a successful commercialisation.Present production processes rely mostly upon extraction of resveratrol,either from the skin of grape berries, or from Knot weed. This is alabour intensive process and generates low yield which, therefore,prompts an incentive for the development of novel, more efficient andhigh-yielding production processes.

In plants, the phenylpropanoid pathway is responsible for the synthesisof a wide variety of secondary metabolic compounds, including lignins,salicylates, coumarins, hydroxycinnamic amides, pigments, flavonoids andphytoalexins. Indeed formation of resveratrol in plants proceeds throughthe phenylpropanoid pathway. The amino acid L-phenylalanine is convertedinto trans-cinnamic acid through the non-oxidative deamination byL-phenylalanine ammonia lyase (PAL) (FIG. 2). Next, trans-cinnamic acidis hydroxylated at the para-position to 4-coumaric acid(4-hydroxycinnamic acid) by cinnamate-4-hydroxylase (C4H), a cytochromeP450 monooxygenase enzyme, in conjunction with NADPH:cytochrome P450reductase (CPR). The 4-coumaric acid, is subsequently activated to4-coumaroyl-CoA by the action of 4-coumarate-CoA ligase (4CL). Finally,resveratrol synthase (VST) catalyses the condensation of a phenylpropaneunit of 4-coumaroyl-CoA with malonyl CoA, resulting in formation ofresveratrol.

Recently, a yeast was disclosed that could produce resveratrol from4-coumaric acid that is found in small quantities in grape must (Beckeret al. 2003). The production of 4-coumaroyl-CoA, and concomitantresveratrol, in laboratory strains of S. cerevisiae, was achieved byco-expressing a heterologous coenzyme-A ligase gene, from hybrid poplar,together with the grapevine resveratrol synthase gene (vst1). The othersubstrate for resveratrol synthase, malonyl-CoA, is already endogenouslyproduced in yeast and is involved in de novo fatty-acid biosynthesis.The study showed that cells of S. cerevisiae could produce minuteamounts of resveratrol, either in the free form or in theglucoside-bound form, when cultured in synthetic media that wassupplemented with 4-coumaric acid.

However, said yeast would not be suitable for a commercial applicationbecause it suffers from low resveratrol yield, and requires addition of4-coumaric acid, which is only present in few industrial media. In orderto facilitate and broaden the application of resveratrol as both apharmaceutical and neutraceutical, it is therefore highly desirable toobtain a yeast that can produce resveratrol directly from glucose,without addition of 4-coumaric acid.

A recent study (Ro and Douglas, 2004) describes the reconstitution ofthe entry point of the phenylpropanoid pathway in S. cerevisiae byintroducing PAL, C4H and CPR from Poplar. The purpose was to evaluatewhether multienzyme complexes (MECs) containing PAL and C4H arefunctionally important at this entry point into phenylpropanoidmetabolism. By feeding the recombinant yeast with [3H]-phenylalanine itwas found that the majority of metabolized [3H]-phenylalanine wasincorporated into 4-[3H]-coumaric acid, and that phenylalaninemetabolism was highly reduced by inhibiting C4H activity. Moreover,PAL-alone expressers metabolized very little phenylalanine into cinnamicacid. When feeding [3H]-phenylalanine and [14C]-trans-cinnamic acidsimultaneously to the triple expressers, no evidence was found forchanneling of the endogenously synthesized [3H]-trans-cinnamic acid into4-coumaric acid. Therefore, efficient carbon flux from phenylalanine to4-coumaric acid via reactions catalyzed by PAL and C4H does not appearto require channeling through a MEC in yeast, and sheer biochemicalcoupling of PAL and C4H seems to be sufficient to drive carbon flux intothe phenylpropanoid pathway. In yet another study (Hwang et al., 2003)production of plant-specific flavanones by Escherichia coli was achievedthrough expression of an artificial gene cluster that contained threegenes of a phenyl propanoid pathway of various heterologous origins; PALfrom the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomycescoelicolor, and chalcone synthase (CHS) from the licorice plantGlycyrrhiza echinata. These pathways bypassed C4H, because the bacterial4CL enzyme ligated coenzyme A to both trans-cinnamic acid and 4-coumaricacid. In addition, the PAL from Rhodotorula rubra uses bothphenylalanine and tyrosine as the substrates. Therefore, E. coli cellscontaining the gene clusters and grown on glucose, produced smallamounts of two flavanones, pinocembrin (0.29 g/l) from phenylalanine andnaringenin (0.17 g/l) from tyrosine. In addition, large amounts of theirprecursors, 4-coumaric acid and trans-cinnamic acid (0.47 and 1.23mg/liter respectively), were acumulated. Moreover, the yields of thesecompounds could be increased by addition of phenylalanine and tyrosine.

Whereas the enzyme from dicotylic plants utilizes only phenylalanineefficiently, several studies indicated that PAL from monocotylic plants,and some micro-organisms, utilizes tyrosine as well (Rösler et al.,1997). In such reactions the enzyme activity is designated tyrosineammonia lyase (TAL, FIG. 3). Conversion of tyrosine by TAL results inthe direct formation of 4-coumaric acid without the intermediacy of C4Hand CPR. Mostly both activities reside on the same polypeptide and havevery similar catalytic efficiencies, in spite of large differences in Kmand turnover number. However, most PAL/TAL enzymes from plants preferphenylalanine rather than tyrosine. The level of TAL activity is mostlylower than PAL activity, but the magnitude of this difference variesover a wide range. For example, the parsley enzyme has a Km forphenylalanine of 15-25 μM and for tyrosine 2.0-8.0 mM with turnovernumbers 22 s⁻¹ and 0.3 s⁻¹ respectively. In contrast, the maize enzymehas a Km for phenylalanine only 15-fold higher than for tyrosine, andturnover numbers about 10-fold higher. Moreover, in the red yeasts,Rhodotorula glutinis (Rhodosporidium toruloides) and -rubra, the TALcatalytic activity is close to the PAL catalytic activity with a ratioof TAL/PAL of approximately 0.58. It is believed that the PAL enzyme inthese yeasts degrades phenylalanine as a catabolic function and thetrans-cinnamic acid formed is converted to benzoate and other cellularmaterials, whereas in plants it is thought to be merely a regulatoryenzyme in the biosynthesis of lignin, isoflavonoids and otherphenylpropanoids.

Recently, an open reading frame was found in the bacterium Rhodobactercapsulatus that encodes a hypothetical biosynthetic tyrosine ammonialyase (TAL) that is involved in the biosynthesis of the chromophore ofthe photoactive yellow protein (Kyndt et al., 2002). This was the firsttime that a PAL-homologous gene was found in bacteria. The TAL gene wasisolated and overproduced in Escherichia coli. The Km and kcat valuesfor the conversion of tyrosine to 4-coumaric acid were 15.6 μM and 27.7s⁻¹ respectively, and for conversion of L-phenylalanine totrans-cinnamic acid were 1277 μM and 15.1 s⁻¹ respectively. As aconsequence of the smaller Km and a slightly larger kcat, the enzymeshows a strong preference for tyrosine over L-phenylalanine, with acatalytic efficiency (Km/kcat) for tyrosine of approximately 150-foldlarger than for phenylalanine. The kinetic studies established thattyrosine, and not L-phenylalanine, is the natural substrate of theenzyme under physiological conditions. Very recently a study describedthe heterologous coexpression of phenylalanine ammonia lyase,cinnamate-4-hydroxylase, 4-coumarate-Coa-ligase and chalcone synthase,for the production of flavonoids in E. coli (Watts et al., 2004). Thesimultaneous expression of all four genes, however, was not successfulbecause of a nonfunctional cinnamate-4-hydroxylase. The substitution ofphenylalanine ammonia lyase and cinnamate-4-hydroxylase by a newtyrosine ammonia lyase that was cloned from Rhodobacter sphaeroides,could, however, solved the problem and led to high-level production ofthe flavonone naringenin. Furthermore, said tyrosine ammonia lyase fromRhodobacter sphaeroides is also used for heterologous production of4-coumaric acid (i.e. para-hydroxycinnamic acid) in Escherichia coli(US-A-2004059103). Evenmore, further methods for development of abiocatalyst for conversion of glucose into 4-coumaric acid aredescribed. US-A-2004023357 discloses a tyrosine ammonia lyase from theyeast Trichosporon cutaneum for the production of coumaric acid inEscherichia coli and Saccharomyces cerevisiae. US-A-2001053847 describesthe incorporation of the wild type PAL from the yeast Rhodotorulaglutinis into E. coli, underlining the ability of the wildtype PAL toconvert tyrosine directly to 4-coumaric acid. Moreover, there is alsoexemplification of incorporation of the wildtype PAL from the yeastRhodotorula glutinis, plus a plant C4H and CPR into E. coli and S.cerevisiae. Also described is the development of a biocatalyst throughmutagenesis of the wild type yeast PAL Rhodotorula glutinis withenhanced TAL activity (US-A-6521748). Neither of the aforementionedpatents claim the incorporation of 4CL and VST for the production ofresveratrol.

Recently, evidence was shown that the filamentous fungi A. oryzaecontained the enzyme chalcone synthase (CHS) that is normally involvedin the biosynthesis of flavonoids, such as naringenin, in plants(Seshime et al., 2005). Indeed it was also shown that A. oryzaecontained the major set of genes responsible forphenylpropanoid-flavonoid metabolism, i.e PAL, C4H and 4CL. However,there is no evidence that A. oryzae contained a stilbene synthase suchas resveratrol synthase.

The present invention now provides a micro-organism having an operativemetabolic pathway comprising at least one enzyme activity, said pathwayproducing 4-coumaric acid and producing resveratrol therefrom or anoligomeric or glycosidically-bound derivative thereof. Such amicro-organism may be naturally occurring and may be isolated bysuitable screening procedures, but more preferably is geneticallyengineered.

Preferably, said resveratrol or derivative is produced in a reactioncatalysed by an enzyme in which endogenous malonyl-CoA is a substrate,and preferably said resveratrol is produced from 4-coumaroyl-CoA.

Said resveratrol or derivative is preferably produced from4-coumaroyl-CoA by a resveratrol synthase which is preferably expressedin said micro-organism from nucleic acid coding for said enzyme which isnot native to the micro-organism.

Generally herein, unless the context implies otherwise, references toresveratrol include reference to oligomeric or glycosidically boundderivatives thereof, including particularly piceid.

Thus, in certain preferred embodiments, said resveratrol synthase is aresveratrol synthase (EC 2.3.1.95) from a plant belonging to the genusof Arachis, e.g. A. glabatra, A. hypogaea, a plant belonging to thegenus of Rheum, e.g. R. tataricum, a plant belonging to the genus ofVitus, e.g. V. labrusca, V. riparaia, V. vinifera, or any one of thegenera Pinus, Piceea, Lilium, Eucalyptus, Parthenocissus, Cissus,Calochortus, Polygonum, Gnetum, Artocarpus, Nothofagus, Phoenix,Festuca, Carex, Veratrum, Bauhinia or Pterolobium.

Preferably, said 4-coumaric acid is produced from trans-cinnamic acid,suitably by an enzyme in a reaction catalysed by said enzyme in whichoxygen is a substrate, NADH or NADPH is a cofactor and NAD⁺ or NADP⁺ isa product.

Thus, said 4-coumaric acid may be produced from trans-cinnamic acid by acinnamate 4-hydroxylase, which preferably is expressed in saidmicro-organism from nucleic acid coding for said enzyme which is notnative to the micro-organism.

In certain preferred embodiments, including those referred to in theparagraphs above, said cinnamate-4-hydroxylase is acinnamate-4-hydroxylase (EC 1.14.13.11) from a plant or amicro-organism. The plant may belong to the genus of Arabidopsis, e.g.A. thaliana, a plant belonging to the genus of Citrus, e.g. C. sinensis,C.×paradisi, a plant belonging to the genus of Phaseolus, e.g. P.vulgaris, a plant belonging to the genus of Pinus, e.g. P. taeda, aplant belonging to the genus of Populus, e.g. P. deltoides, P.tremuloides, P. trichocarpa, a plant belonging to the genus of Solanum,e.g. S. tuberosum, a plant belonging to the genus of Vitus, e.g. Vitusvinifera, a plant belonging to the genus of Zea, e.g. Z. mays, or otherplant genera e.g. Ammi, Avicennia, Camellia, Camptotheca, Catharanthus,Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta,Saccharum, Vigna. The micro-organism might be a fungus belonging to thegenus Aspergillus, e.g. A. oryzae.

Preferably, said 4-coumaric acid is produced from tyrosine in a reactioncatalysed by an enzyme in which ammonia is produced and suitably, said4-coumaric acid is produced from tyrosine by a L-phenylalanine ammonialyase or a tyrosine ammonia lyase, e.g. tyrosine ammonia lyase (EC4.3.1.5) from yeast or bacteria. Suitably, the tyrosine ammonia lyase isfrom the yeast Rhodotorula rubra or from the bacterium Rhodobactercapsulatus.

Optionally, said tyrosine ammonia lyase is expressed in saidmicro-organism from nucleic acid coding for said enzyme which is notnative to the micro-organism.

Alternatively, said trans-cinnamic acid may be produced fromL-phenylalanine in a reaction catalysed by an enzyme in which ammonia isproduced and suitably said trans-cinnamic acid is formed fromL-phenylalanine by a phenylalanine ammonia lyase.

In certain preferred embodiments, said L-phenylalanine ammonia lyase isa L-phenylalanine ammonia lyase (EC 4.3.1.5) from a plant or amicro-organism. The plant may belong to the genus of Arabidopsis, e.g.A. thaliana, a plant belonging to the genus of Brassica, e.g. B. napus,B. rapa, a plant belonging to the genus of Citrus, e.g. C. reticulata,C. clementinus, C. limon, a plant belonging to the genus of Phaseolus,e.g. P. coccineus, P. vulgaris, a plant belonging to the genus of Pinus,e.g. P. banksiana, P. monticola, P. pinaster, P. sylvestris, P. taeda, aplant belonging to the genus of Populus, e.g. P. balsamifera, P.deltoides, P. Canadensis, P. kitakamiensis, P. tremuloides, a plantbelonging to the genus of Solanum, e.g. S. tuberosum, a plant belongingto the genus of Prunus, e.g. P. avium, P. persica, a plant belonging tothe genus of Vitus, e.g. Vitus vinifera, a plant belonging to the genusof Zea, e.g. Z. mays or other plant genera e.g. Agastache, Ananas,Asparagus, Bromheadia, Bambusa, Beta, Betula, Cucumis, Camellia,Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita,Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus,Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca,Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot, Medicago,Mesembryanthemum, Nicotiana, Olea, Oryza, Pisum, Persea, Petroselinum,Phalaenopsis, Phyllostachys, Physcomitrella, Picea, Pyrus, Quercus,Raphanus, Rehmannia, Rubus, Sorghum, Sphenostylis, Stellaria,Stylosanthes, Triticum, Trifolium, Triticum, Vaccinium, Vigna, Zinnia.The micro-organism might be a fungus belonging to the genus Agaricus,e.g. A. bisporus, a fungus belonging to the genus Aspergillus, e.g. A.oryzae, A. nidulans, A. fumigatus, a fungus belonging to the genusUstilago, e.g. U. maydis, a bacterium belonging to the genusRhodobacter, e.g. R. capsulatus, a yeast belonging to the genusRhodotorula, e.g. R. rubra.

Suitably, said L-phenylalanine ammonia lyase is expressed in saidmicro-organism from nucleic acid coding for said enzyme which is notnative to the micro-organism.

Preferably, 4-coumaroyl-CoA is formed in a reaction catalysed by anenzyme in which ATP and CoA are substrates and ADP is a product andsuitably 4-coumaroyl-CoA is formed in a reaction catalysed by a4-coumarate-CoA ligase.

Said 4-coumarate-CoA ligase may be a 4-coumarate-CoA ligase (EC6.2.1.12) from a plant, a micro-organism or a nematode. The plant maybelong to the genus of Abies, e.g. A. beshanzuensis, B. firma, B.holophylla, a plant belonging to the genus of Arabidopsis, e.g. A.thaliana, a plant belonging to the genus of Brassica, e.g. B. napus, B.rapa, B. oleracea, a plant belonging to the genus of Citrus, e.g. C.sinensis, a plant belonging to the genus of Larix, e.g. L. decidua, L.gmelinii, L. griffithiana, L. himalaica, L. kaempferi, L. laricina, L.mastersiana, L. occidentalis, L. potaninii, L. sibirica, L. speciosa, aplant belonging to the genus of Phaseolus, e.g. P. acutifolius, P.coccineus, a plant belonging to the genus of Pinus, e.g. P. armandii P.banksiana, P. pinaster, a plant belonging to the genus of Populus, e.g.P. balsamifera, P. tomentosa, P. tremuloides, a plant belonging to thegenus of Solanum, e.g. S. tuberosum, a plant belonging to the genus ofVitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g.Z. mays, or other plant genera e.g. Agastache, Amorpha, Cathaya, Cedrus,Crocus, Festuca, Glycine, Juglans, Keteleeria, Lithospermum, Lolium,Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana,Nothotsuga, Oryza, Pelargonium, Petroselinum, Physcomitrella, Picea,Prunus, Pseudolarix, Pseudotsuga, Rosa, Rubus, Ryza, Saccharum, Suaeda,Thellungiella, Triticum, Tsuga. The micro-organism might be afilamentous fungi belonging to the genus Aspergillus, e.g. A. flavus, A.nidulans, A. oryzae, A. fumigatus, a filamentous fungus belonging to thegenus Neurospora, e.g. N. crassa, a fungus belonging to the genusYarrowia, e.g. Y. lipolytica, a fungus belonging to the genus ofMycosphaerella, e.g. M. graminicola, a bacterium belonging to the genusof Mycobacterium, e.g. M. bovis, M. leprae, M. tuberculosis, a bacteriumbelonging to the genus of Neisseria, e.g. N. meningitidis, a bacteriumbelonging to the genus of Streptomyces, e.g. S. coelicolor, a bacteriumbelonging to the genus of Rhodobacter, e.g. R. capsulatus, a nematodebelonging to the genus Ancylostoma, e.g. A. ceylanicum, a nematodebelonging to the genus Caenorhabditis, e.g. C. elegans, a nematodebelonging to the genus Haemonchus, e.g. H. contortus, a nematodebelonging to the genus Lumbricus, e.g. L. rubellus, a nematode belongingto the genus Meilodogyne, e.g. M. hapla, a nematode belonging to thegenus Strongyloidus, e.g. S. rattii, S. stercoralis, a nematodebelonging to the genus Pristionchus, e.g. P. pacificus.

Optionally, a NADPH:cytochrome P450 reductase (CPR) has beenrecombinantly introduced into said micro-organism. This may be a plantCPR introduced into a non-plant micro-organism. Alternatively, a nativeNADPH:cytochrome P450 reductase (CPR) has been overexpressed in saidmicro-organism.

In certain preferred embodiments, including those referred to in theparagraphs above, said NADPH:cytochrome P450 reductase is aNADPH:cytochrome P450 reductase (EC 1.6.2.4) from a plant belonging tothe genus of Arabidopsis, e.g. A. thaliana, a plant belonging to thegenus of Citrus, e.g. C. sinensis, C.×paradisi, a plant belonging to thegenus of Phaseolus, e.g. P. vulgaris, a plant belonging to the genus ofPinus, e.g. P. taeda, a plant belonging to the genus of Populus, e.g. P.deltoides, P. tremuloides, P. trichocarpa, a plant belonging to thegenus of Solanum, e.g. S. tuberosum, a plant belonging to the genus ofVitus, e.g. Vitus vinifera, a plant belonging to the genus of Zea, e.g.Z. mays, or other plant genera e.g. Ammi, Avicennia, Camellia,Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum,Physcomitrella, Ruta, Saccharum, Vigna.

Whilst the micro-organism may be naturally occurring, preferably atleast one copy of at least one genetic sequence encoding a respectiveenzyme in said metabolic pathway has been recombinantly introduced intosaid micro-organism.

Additionally or alternatively to introducing coding sequences coding fora said enzyme, one may provide one or more expression signals, such aspromoter sequences, not natively associated with said coding sequence insaid organism. Thus, optionally, at least one copy of a genetic sequenceencoding a tyrosine ammonia lyase is operatively linked to an expressionsignal not natively associated with said genetic sequence in saidorganism, and/or at least one copy of a genetic sequence encoding aL-phenylalanine ammonia lyase is operatively linked to an expressionsignal not natively associated with said genetic sequence in saidorganism.

Optionally, at least one copy of a genetic sequence encoding cinnamate4-hydroxylase, whether native or not, is operatively linked to anexpression signal not natively associated with said genetic sequence insaid organism.

Optionally, at least one copy of a genetic sequence encoding a4-coumarate-CoA ligase, whether native or not, is operatively linked toan expression signal not natively associated with said genetic sequencein said organism.

Optionally, at least one copy of a genetic sequence encoding aresveratrol synthase, whether native or not, is operatively linked to anexpression signal not natively associated with said genetic sequence insaid organism.

Expression signals include nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Such sequences may include promoters, translation leadersequences, introns, and polyadenylation recognition sequences.

In certain aspects the invention provides a metabolically engineeredmicro-organism having an operative metabolic pathway in which a firstmetabolite is transformed into a second metabolite in a reactioncatalysed by a first enzyme, said reaction step producing ammonia, andin which said second metabolite is transformed into a third metabolitein a reaction catalysed by a second enzyme, in which oxygen is asubstrate, NADPH or NADH is a cofactor and NADP⁺ or NAD⁺ is a product,and in which said third metabolite is transformed into a fourthmetabolite in a reaction catalysed by a third enzyme in which ATP andCoA is a substrate, and ADP is a product, and in which said fourthmetabolite is transformed into a fifth metabolite in a reactioncatalysed by a fourth enzyme in which endogenous malonyl-CoA is asubstrate.

The present invention also provides a metabolically engineeredmicro-organism having an operative metabolic pathway in which a firstmetabolite is transformed into a said third metabolite catalysed by afirst enzyme, said reaction step producing ammonia, without theinvolvement of said second enzyme, and in which said third metabolite istransformed into a said fourth metabolite in a reaction catalysed by asaid third enzyme in which ATP and CoA is a substrate, and ADP is aproduct, and in which said fourth metabolite is transformed into a saidfifth metabolite in a reaction catalysed by a said fourth enzyme inwhich endogenous malonyl-CoA is a substrate.

The micro-organisms described above include ones containing one or morecopies of an heterologous DNA sequence encoding phenylalanine ammonialyase operatively associated with an expression signal, and containingone or more copies of an heterologous DNA sequence encodingcinnamate-4-hydroxylase operatively associated with an expressionsignal, and containing one or more copies of an heterologous DNAsequence encoding 4-coumarate-CoA-ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal.

They include also ones lacking cinnamate-4-hydroxylase activity, andcontaining one or more copies of a heterologous DNA sequence encodingtyrosine ammonia lyase operatively associated with an expression signal,and containing one or more copies of an heterologous DNA sequenceencoding 4-coumarate-CoA-ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal.

In the present context the term “micro-organism” relates to microscopicorganisms, including bacteria, microscopic fungi, including yeast.

More specifically, the micro-organism may be a fungus, and morespecifically a filamentous fungus belonging to the genus of Aspergillus,e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging tothe genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus,S. exiguus, S. sevazzi, S. uvarum, a yeast belonging to the genusKluyveromyces, e.g. K. lactis K. marxianus var. marxianus, K.thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilisC. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeastbelonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P.sorbitophila, or other yeast genera, e.g. Cryptococcus, Debaromyces,Hansenula, Pichia, Yarrowia, Zygosaccharomyces or Schizosaccharomyces.Concerning other micro-organisms a non-exhaustive list of suitablefilamentous fungi is supplied: a species belonging to the genusPenicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor,Mortierella, Trichoderma.

Concerning bacteria a non-exhaustive list of suitable bacteria is givenas follows: a species belonging to the genus Bacillus, a speciesbelonging to the genus Escherichia, a species belonging to the genusLactobacillus, a species belonging to the genus Lactococcus, a speciesbelonging to the genus Corynebacterium, a species belonging to the genusAcetobacter, a species belonging to the genus Acinetobacter, a speciesbelonging to the genus Pseudomonas, etc.

The preferred micro-organisms of the invention may be S. cerevisiae, A.niger, A. oryzae, E. coli, L. lactis or B. subtilis.

The constructed and engineered micro-organism can be cultivated usingcommonly known processes, including chemostat, batch, fed-batchcultivations, etc.

Thus, the invention includes a method for producing resveratrol or anoligomeric or glycosidically-bound derivative thereof comprisingcontacting a non-plant cell with a carbon substrate in the substantialabsence of an external source of 4-coumaric acid, said cell having thecapacity to produce resveratrol or an oligomeric or glycosidically-boundderivative thereof under the conditions, in which the micro-organism maybe selected from the group consisting of fungi and bacteria, especiallyyeast.

Said carbon substrate is optionally selected from the group offermentable carbon substrates consisting of monosaccharides,oligosaccharides and polysaccharides, e.g. glucose, fructose, galactose,xylose, arabinose, mannose, sucrose, lactose, erythrose, threose, and/orribose. Said carbon substrate may additionally or alternatively beselected from the group of non-fermentable carbon substrates includingethanol, acetate, glycerol, and/or lactate. Said non-fermentable carbonsubstrate may additionally or alternatively be selected from the groupof amino acids and may be phenylalanine and/or tyrosine.

In an alternative aspect, the invention includes a method for producingresveratrol or an oligomeric or glycosidically-bound derivative thereofthrough heterologous expression of nucleotide sequences encodingphenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate-CoAligase and resveratrol synthase and also a method for producingresveratrol through heterologous expression of nucleotide sequencesencoding tyrosine ammonia lyase, 4-coumarate-CoA ligase and resveratrolsynthase.

Resveratrol or an oligomeric or glycosidically-bound derivative thereofso produced may be used as a nutraceutical in a dairy product or abeverage such as beer.

Resveratrol produced according to the invention may be cis-resveratrolor trans-resveratrol, but it is to be expected that the trans-form willnormally predominate.

BRIEF DESCRIPTION OF THE DRAWINGS

To assist in the ready understanding of the above description of theinvention reference has been made to the accompanying drawings in which:

FIG. 1 shows the chemical structure of trans-resveratrol;

FIG. 2 shows the phenylpropanoid pathway utilising phenylalanine ammonialyase acting on L-phenylalanine; and

FIG. 3 shows the alternative pathway utilising phenylalanine ammonialyase acting on L-tyrosine.

FIG. 4 shows the HPLC-chromatograms of extracts of S. cerevisiae strainsFSSC-PALC4H4CLVST, FSSC-TAL4CLVST, grown on 100 g/l galactose. Achromatogram of 60 nanogram of pure resveratrol is included.

FIG. 5 shows the UV absorption spectrum for pure trans-resveratrol andtrans-resveratrol produced by S. cerevisiae strain FSSC-PALC4H4CLVST,grown on 100 g/l galactose.

FIG. 6 shows the HPLC-chromatograms of extracts from E. coli strainsFSEC-TAL4CLVST and FSEC-control, grown on 50 g/l glucose.

FIG. 7 shows the HPLC-chromatograms of extracts from E. coli strainsFSEC-TAL4CLVST and FSEC-control, grown on 50 g/l glucose with additionof 20 mg/l coumaric acid. The UV absorption spectrum fortrans-resveratrol produced in strain FSEC-TAL4CLVST is included.

The invention will be further described and illustrated by the followingnon-limiting examples.

EXAMPLES Example 1 Isolation of Genes Encoding PAL, TAL, C4H, CPR, 4CL,and VST

Phenylalanine ammonia lyase (PAL2) (Cochrane et al., 2004; SEQ ID NO: 1,2), cinnamate 4-hydroxylase (C4H) (Mizutani et al., 1997; SEQ ID NO: 3,4) and 4-coumarate:CoenzymeA ligase (4CL1) (Hamberger and Hahlbrock2004; Ehlting et al., 1999; SEQ ID NO: 5, 6) were isolated via PCR fromA. thaliana cDNA (BioCat, Heidelberg, Germany) using the primers intable 1. PAL2 and 4CL1 were chosen amongst several A. thalianahomologues due to favourable kinetic parameters towards cinnamic acidand coumaroyl-CoA, respectively (Cochrane et al., 2004; Hamberger andHahlbrock 2004; Ehlting et al., 1999).

The coding sequence of resveratrol synthase (VST) from Rheumtataricum(Samappito et al., 2003; SEQ ID NO: 7, 8) and tyrosine ammonialyase (TAL) from Rhodobacter capsulatus (Kyndt et al., 2002; SEQ ID NO:11, 12) were codon optimized for expression in S. cerevisiaeusing theonline service backtranslation tool at www DOT entelechon.com (“.”replaced with “DOT” to inactivate URL), yielding sequence SEQ ID NO: 9,10and SEQ ID NO: 13, 14 respectively. Oligos for the synthetic geneassembly were constructed at MWG Biotech and the synthetic gene wasassembled by PCR using a slightly modified method protocol of fromMartin et al. (2003) described below.

TABLE 1 Primers and restriction sites for the amplification of genesPrimer for amplification of gene* Restriction Restriction (Restrictionsites are underlined) Gene site: primer site: vector5′-CGGAATTCTCATGGATCAAATCGAAGCAATGTT PAL2 EcoR1 EcoR15′-CGACTAGTTTAGCAAATCGGAATCGGAGC PAL2 Spe1 Spe1 5′-CGCTCGAGATATGGACCTCCTCTTGCTGGA C4H Xho1 Xho1 5′-CGGGTACCTTAACAGTTCCTTGGTTTCATAACC4H Kpnl Kpn1 5′-GCTCTAGACCT ATGGCGCCACAAGAACAAGCAGTTT 4CL1 Xba1 Spe15′-GCGGATCCCCT TCACAATCCATTTGCTAGTTT TGCC 4CL1 BamH1 BglII5′-CCGGATCCAAATGGCCCCAGAAGAGAGCAGG VST BamH1 BamH1 5′-CGCTCGAGTTAAGTGATCAATGGAACCGAAGACAG VST Xho1 Xho1 5′-CCGAATTCCCATGACCCTGCAATCTCAAACAGCTAAAG TAL EcoR1 EcoR15′-CCACTAGTTTAAGCAGGTGGATCGGCAGCT TAL Spe1 Spe15′-CCCTCGAGATCATGCCGTTTGGAATAGACAACACCGA CPR1 Xho1 Xho15′-CCAAGCTTATCGGGCTGATTACCAGACATCTTCTTG CPR1 HindIII HindIII5′-CCGGATCCCCATGTCCTCTTCTTCTTCTTCGTCAAC AR2 Bamh1 Bamh15′-CCCTCGAGGTGAGTGTGTGGCTTCAATAGTTT CG AR2 Xho1 Xho1 *SEQ ID Nos 19-32

Primers from MWG for the assembly of the synthetic gene were dissolvedin milliQ-water to a concentration of 100 pmole/μl. An aliquot of 5 μlof each primer was combined in a totalmix and then diluted 10-fold withmilliQ water. The gene was assembled via PCR using 5 μl diluted totalmixper 50 μl as template for fusion DNA polymerase (Finnzymes). The PCRprogramme was as follows: Initial 98° C. for 30 s., and then 30 cycleswith 98° C. for 10 s., 40° C. for 1 min. and 72° C. at 1 min./1000basepairs, and a final 72° C. for 5 min. From the resulting PCRreaction, 20 μl was purified on 1% agarose gel. The result was a PCRsmear and the regions around the wanted size were cut out from agarosegel and purified using the QiaQuick Gel Extraction Kit (Qiagen). A finalPCR with the outer primers (for TAL and VST) in table 1 rendered therequired TAL and VST genes. Point mutations were corrected using eitherthe Quickchange site directed mutagenesis II kit (Stratagene, La Jolla,Calif.), or using PCR from overlapping error free DNA stretches fromseveral different E. coli subclones.

NADPH:Cytochrome P450 reductase (CPR) from A. thaliana (AR2) (Mizutaniand Ohta, 1998; SEQ ID NO: 17, 18) and from S. cerevisiae (CPR1) (Aoyamaet al., 1978; SEQ ID NO: 15, 16), were isolated from A. thaliana cDNA(BioCat, Heidelberg, Germany) and S. cerevisae genomic DNA,respectively, using the primers in table 1.

Example 2 Construction of a Yeast Vector for Expression of PAL

The gene encoding PAL, isolated as described in example 1, wasreamplified by PCR using forward- and reverse primers, with 5′ overhangscontaining EcoR1 and Spe1 restriction sites (table 1). The amplified PALPCR product was digested with EcoR1/Spe1 and ligated into EcoR1/Spe1digested pESC-URA vector (Stratagene), resulting in vector pESC-URA-PAL.The sequence of the gene was verified by sequencing of two differentclones.

Example 3 Construction of a Yeast Vector for Expression of PAL and C4H

The gene encoding C4H, isolated as described in example 1, was amplifiedby PCR using the forward- and reverse primers, with 5′ overhangscontaining Xho1 and Kpn1 restriction sites. The amplified C4HPCR-product was digested with Xho1/Kpn1 and ligated into similarlydigested pESC-URA-PAL vector. The resulting plasmid, pESC-URA-PAL-C4H,contained the genes encoding PAL and C4H under the control of thedivergent GAL1/GAL10 promoter. The sequence of the gene encoding C4H wasverified by sequencing of two different clones.

Example 4 Construction of a Yeast Vector for Expression of 4CL

The gene encoding 4CL was isolated as described in example 1. Theamplified 4CL PCR-product was digested with Xba1/BamH1 and ligated intoSpe1/BglII digested pESC-TRP vector (Stratagene), resulting in vectorpESC-TRP-4CL. Two different clones of pESC-TRP-4CL were sequenced toverify the sequence of the cloned gene.

Example 5 Construction of a Yeast Vector for Expression of 4CL and VST

The gene encoding VST was isolated as described in example 1. Theamplified synthetic VST gene was digested with BamH1/Xho1 and ligatedinto BamH1/Xho1 digested pESC-TRP-4CL (example 4). The resultingplasmid, pESC-TRP-4CL-VST, contained the genes encoding 4CL and VSTunder the control of the divergent GAL1/GAL10 promoter. The sequence ofthe gene encoding VST was verified by sequencing of two different clonesof pESC-TRP-4CL-VST.

Example 6 Construction of a Yeast Vector for Expression of TAL

The gene encoding TAL was isolated as described in example 1. Theamplified synthetic TAL gene was digested with EcoR1/Spe1 and ligatedinto EcoR1/Spe1-digested pESC-URA vector. The resulting plasmid,pESC-URA-TAL, contained the gene encoding for TAL under the control ofthe divergent GAL1/GAL10 promoter. The sequence was verified bysequencing of two different clones of pESC-URA-TAL.

Example 7 Construction of a Yeast Vector for Overexpression of S.cerevisiae Endogenous CPR

The gene encoding CPR from S. cerevisiae (CPR1) was isolated asdescribed in example 1. The amplified CPR1 gene was digested withXho1/HindIII and ligated into Xho1/HindIII-digested pESC-LEU vector(Stratagene), resulting in vector pESC-LEU-CPR1. The sequence wasverified by sequencing of two different clones of pESC-LEU-CPR1.

Example 8 Construction of a Yeast Vector for Overexpression of A.thaliana CPR (AR2)

The gene encoding CPR from A. thaliana (AR2) was isolated as describedin example 1. The amplified AR2 gene was digested with BamH1/Xho1 andligated into BamH1/Xho1 digested pESC-LEU vector (Stratagene), resultingin vector pESC-LEU-AR2. The sequence was verified by sequencing of twodifferent clones of pESC-LEU-AR2.

Example 9 Expression of the Pathway to Resveratrol in the Yeast S.cerevisiae Using PAL, C4H, 4CL and VST

Yeast strains containing the appropriate genetic markers weretransformed with the vectors described in examples 2, 3, 4, 5, 6, 7 and8, separately or in combination. The transformation of the yeast cellwas conducted in accordance with methods known in the art, for instance,by using competent cells or by electroporation (see, e.g., Sambrook etal., 1989). Transformants were selected on medium lacking uracil and/ortryptophan and streak purified on the same medium.

S. cerevisiae strain CEN.PK 113-5D (MATa ura3) was transformedseparately with the vector pESC-URA-PAL (example 2), yielding the strainFSSC-PAL, and with pESC-URA-PAL-C4H (example 3), resulting in the strainFSSC-PALC4H. S. cerevisiae strain FS01267 (MATa trp1 ura3) wasco-transformed with pESC-URA-PAL-C4H and pESC-TRP-4CL (example 4), andthe transformed strain was named FSSC-PALC4H4CL. The same strain wasalso co-transformed with pESC-URA-PAL-C4H and pESC-TRP-4CL-VST (example5), resulting in the strain FSSC-PALC4H4CLVST.

Example 10 Expression of the Pathway to Resveratrol in S. cerevisiaeUsing TAL, 4CL and VST

S. cerevisiae strain CEN.PK 113-5D (MATa ura3) was transformedseparately with the vector pESC-URA-TAL (example 6), yielding the strainFSSC-TAL. S. cerevisiae strain FS01267 (MATa trp1 ura3) wasco-transformed with pESC-URA-TAL (example 6) and pESC-TRP-4CL (example4), and the transformed strain was named FSSC-TAL4CL. The same strainwas also co-transformed with pESC-URA-TAL and pESC-TRP-4CL-VST (example5), resulting in the strain FSSC-TAL4CLVST. Transformants were selectedon medium lacking uracil and or tryptophan and streak purified on thesame medium.

Example 11 Expression of the Pathway to Resveratrol in S. cerevisiaewith Overexpressed Endogenous CPR

S. cerevisiae strain FS01277 (MATa ura3 leu2 trp1) was co-transformedwith vectors pESC-URA-PAL-C4H (example 3), pESC-TRP-4CL (example 4), andpESC-LEU-CPR1 (example 7). The transformed strain was namedFSSC-PALC4H4CLVSTCPR. Transformants were selected on medium lackinguracil and/or tryptophan and streak purified on the same medium.

Example 12 Expression of the Pathway to Resveratrol in S. Cerevisiaewith Overexpressed A. Thaliana CPR (AR2)

S. cerevisiae strain FS01277 (MATa ura3 leu2 trp1) was co-transformedwith vectors pESC-URA-PAL-C4H (example 3), pESC-TRP-4CL (example 4), andpESC-LEU-AR2 (example 8). The transformed strain was namedFSSC-PALC4H4CLVSTAR2. Transformants were selected on medium lackinguracil and or tryptophan and streak purified on the same medium.

Example 13 Fermentation with Recombinant Yeast Strains in Shake Flasks

The recombinant yeast strains were inoculated from agar plates with asterile inoculation loop and grown in 200 ml defined mineral medium(Verduyn et al, 1992) that contained vitamins, trace elements, 5 g/lglucose and 40 g/l or 100 g/l galactose. The 500 ml stoppered shakeflasks were incubated for three days at 30° C. and 160 rpm.

Example 14 Extraction of Resveratrol

Cells were harvested by centrifugation 5000 g for 5 minutes. An aliquotof 50 ml of supernatant was extracted once with 20 ml ethyl acetate. Theethyl acetate was freeze dried and the dry product redissolved in 0.7 mlmethanol and filtered into HPLC vials.

The cell pellet from 200 ml medium was dissolved in 1 to 2 ml water anddivided into 3 fastprep tubes and broken with glass beads. The crudeextracts from the three tubes were pooled into 10 ml 100% methanol in a50 ml sartorius tube and extracted on a rotary chamber for 48 hours in adark cold room at 4° C. After 48 hours the cell debris was removed viacentrifugation for 5 min. at 5000 g and the methanol was removed byfreeze-drying overnight. The dried residue was redissolved in 1 mlphosphate-citrate buffer pH 5.4 and 10 units beta-glucosidase fromalmonds was added (Sigma) to release resveratrol from putativelyglucoside-bound forms. The mixture was incubated for three hours at 37°C. and then extracted twice with 1 ml ethyl acetate. The combined ethylacetate was freeze dried and the dry residue was redissolved in 0.7 mlmethanol and filtered into HPLC vials.

Example 15 Analysis of Resveratrol

Thin Layer Chromatography

A method based upon thin layer chromatography that enabled the quickseparation of cinnamic, coumaric and resveratrol on the same TLC-platewas developed for quick screening analysis. An aliquot of 1 ml culturecontaining both cells and supernatant were extracted with 500 microliterethyl acetate and centrifuged for 30 s. at 13000 rpm with amicrocentrifuge. The ethyl acetate was dried and redissolved inmethanol. The extracts were analyzed on Silica G plates (0.2 mm AlugramSIL G/UV₂₅₄, Macherey-Nagel) containing a fluorescent indicator. Themobile phase was a mixture of chloroform, ethyl acetate and formic acid(25:10:1).

HPLC

For quantitative analysis of cinnamic acid, coumaric acid, andresveratrol, samples were subjected to separation by high-performanceliquid chromatography (HPLC) Agilent Series 1100 system (HewlettPackard) prior to uv-diode-array detection at λ=306 nm. A Phenomenex(Torrance, Calif., USA) Luna 3 micrometer C18 (100×2.00 mm) column wasused at 40° C. As mobile phase a gradient of acetonitrile and milliqwater (both containing 50 ppm trifluoroacetic acid) was used at a flowof 0.4 ml/min. The gradient profile was linear from 15% acetonitrile to100% acetonitrile over 20 min. The elution times were approximately 3.4min. for coumaric acid, 5.5 min. for free trans-resveratrol and 6.8 min.for cinnamic acid.

Pure resveratrol standard was purchased from Cayman chemical company,whereas pure coumaric acid and cinnamic acid standards were purchasedfrom and Sigma.

Results

Strains FSSC-PALC4H4CLVST and FSSC-TAL4CLVST, were cultivated on 100 g/lgalactose as described in example 13, and analyzed for their content ofintracellular resveratrol according to example 14 and 15. Additionally,a control strain FSSC-control was included that contained the emptyvectors pESC-URA and pESC-TRP only. The HPLC-analysis showed thatstrains FSSC-PALC4H4CLVST and FSSC-TAL4CLVST contained a component witha retention time of 5.5 min. that was identical to trans-resveratrol(FIG. 4). Said result was confirmed by the UV absorption spectra thatwere similar to the absorption spectrum of pure trans-resveratrol (FIG.5) as well, with a λ_(max) of approximately 306 nm.

The results, therefore, demonstrated the presence of an activephenyl-propanoid pathway in S. cerevisiae that led to in vivo productionof trans-resveratrol. The production of resveratrol can most likely beimproved by cultivating the strains under well-defined growth conditionsin batch- and continuous cultures, and/or optimizing theexpression/activities of the individual enzymes.

Example 16 Construction of a Bacterial Vector for Expression of TAL inEscherichia coli

The gene encoding TAL, isolated as described in Example 1, wasreamplified by PCR from the plasmid pESC-URA-TAL (example 6) using theforward primer 5′-CCG CTCGAG CGG ATG ACC CTG CAA TCT CAA ACA GCT AAAG-3′ SEQ ID NO 33 and the reverse primer 5′-GC GGATCC TTA AGC AGG TGGATC GGC AGC T-3′ SEQ ID NO 34 with 5′ overhangs containing therestriction sites XhoI and BamHI, respectively. The introduction ofrestriction sites at the 5′ and 3′ ends of the gene allowed ligation ofthe restricted PCR product into a pET16b vector (Novagen), digested withXhoI and BamHI to yield pET16b-TAL. The pET16b vector contained both theampicillin resistance gene, and the T7 promoter. Hence, above procedureresulted in a vector with an antibiotic selection marker that containedthe gene encoding for TAL under the control of the T7 promoter. Thesequence of the gene encoding TAL was verified by sequencing of oneclone of pET16b-TAL.

Example 17 Construction of a Bacterial Vector for Expression of 4CL andVST in Escherichia coli

The gene encoding VST, isolated as described in example 1, was cut outwith the restriction enzymes BamHI and XhoI from the digested plasmidpESC-TRP-4CL-VST (example 5), which contains the genes encoding 4CL andVST. The VST gene was ligated into a pET26b vector (Novagen), containingthe kanamycin resistance gene, digested with BamHI and SalI to yieldpET26b-VST. The restriction enzymes XhoI and SalI have compatible ends,which enabled proper ligation. The pET26b vector contained both thekanamycin resistance gene, and the T7 promoter. Hence, above procedureresulted in a vector with an antibiotic selection marker that containedthe gene encoding for VST under the control of the T7 promoter.

The gene encoding for 4CL, isolated as described in example 1, wasreamplified by PCR from the plasmid pESC-URA-4CL-VST (example 5) usingthe forward primer 5′-TG CCATGG CA ATGGCGCCAC AAGAACAAGC AGTTT-3′ SEQ IDNO 35 and the reverse primer 5′-GC GGATCC CCT TCA CAA TCC ATT TGC TAGTTT TGCC-3′ SEQ ID NO 36 with 5′ overhangs containing the restrictionsites NcoI and BamHI, respectively. The introduction of restrictionsites at the 5′ and 3′ ends of the gene allowed ligation of therestricted PCR product into a pET16b vector (Novagen) digested with NcoIand BamHI. The resulting plasmid, pET16b-4CL, contained the geneencoding for 4CL under the control of the T7 promoter. Both the T7promoter and the gene encoding for 4CL were reamplified as one fragmentby PCR from the plasmid pET16b-4CL using the forward primer 5′-TTGCGGCCGC AAA TCT CGA TCC CGC GAA ATT AAT ACG-3′ SEQ ID NO 37 and thereverse primer 5′-CG CTCGAG CCT TCA CAA TCC ATT TGC TAG TTT TGCC-3′ SEQID NO 38 with 5′ overhangs, containing the restriction sites NotI andXhoI, respectively. The introduction of restriction sites at the 5′ and3′ ends of the DNA fragment allowed ligation of the restricted PCRproduct into the plasmid pET26b-VST that was digested with NotI and XhoIbefore ligation. The resulting plasmid, pET26b-VST-4CL, contained thetwo genes 4CL and VST that each were under control of an individual T7promoter.

Example 18 Expression of the Pathway to Resveratrol in Escherichia coli,Using TAL, 4CL and VST

The transformation of the bacterial cell was conducted in accordancewith methods known in the art, for instance, by using competent cells orby electroporation (see, e.g., Sambrook et al., 1989). The E. colistrain BL21 (DE3) (Novagen) was co-transformed with the two vectorspET16b-TAL (example 16) and pET26b-VST-4CL (Example 17), resulting instrain FSEC-TAL4CLVST. In addition, E. coli strain BL21 (DE3) wasco-transformed with the two empty vectors pET16b (Novagen) and pET26b(Novagen), resulting in strain FSEC-control, which was used as a controlstrain. Transformants were selected on Luria-Bertani (LB) medium with100 μg/ml ampicillin and 60 μg/ml kanamycin.

Example 19 Fermentation with Recombinant Escherichia coli Strains inShake Flasks

Pre-cultures of Escherichia coli BL21 (DE3) were grown in glass tubes at160 rpm and 37° C. in 7 ml of LB medium containing 100 μg/ml ampicillinand 60 μg/ml kanamycin. Exponentially growing precultures were used forinoculation of 500 ml baffled shake flasks that contained 200 ml LBmedium supplemented with 50 g/l glucose, 5 g/l K₂HPO₄, 80 μg/mlampicillin and 50 μg/ml kanamycin, which were incubated at 160 rpm and37° C. After 5 hours, isopropyl β-thiogalactopyranoside (IPTG) was addedat a final concentration of 1 mM, as an inducer of the T7 promoter thatwas in front of each of the three genes TAL, 4CL and VST. After anincubation period of 48 hours at 37° C., the cells were harvested andsubjected to extraction procedures and analysed for the presence ofproduced resveratrol.

Example 20 Extraction and Analysis of Resveratrol in Escherichia coli

Extraction and analysis was performed using the methods as described inexample 14 and 15.

Results

Strain FSEC-TAL4CLVST and FSEC-control, were cultivated on 50 g/lglucose as described in example 19, and analyzed for their content ofintracellular resveratrol according to example 14 and 15. TheHPLC-analysis showed that strain FSEC-TAL4CLVST did contain considerableamounts of a component with a retention time of 3.4 min., which isidentical to coumaric acid (FIG. 6). However, the extract did notcontain a component that eluted at the same time as trans-resveratrol.Said result, therefore, indicated that the tyrosine ammonia lyase (TAL)was active indeed, but did not lead to production of detactable amountsof resveratrol. The lack of resveratrol formation, however, could be theresult of; i) a non-functional coumarate-CoA ligase (4CL); ii) anon-functional resveratrol synthase (VST); iii) too low levels ofcoumaric acid, caused by either non-optimal cultivation conditions, ornon-optimal expression/activity of TAL, or branching of coumaric acidinto other products. To evaluate said hypotheses, the strains were grownon similar media as described in example 19 but now in the presence of20 mg/l of coumaric acid. The subsequent HPLC-analysis of extracts ofFSEC-TAL4CLVST indeed showed a cluster of peaks around the sameretention time as trans-resveratrol, which was not observed in extractsof FS-control (FIG. 6). Indeed, the UV absorption spectrum of the peakwith a retention time of 5.5 min. was similar to the spectrum of puretrans-resveratrol (FIG. 7), whereas no such spectrum could be obtainedfor peaks in the control strain. The results, therefore, stronglysuggest the presence of an active phenylpropanoid pathway in Escherichiacoli, which can lead to production of resveratrol. Most likely theproduction of resveratrol without addition of coumaric acid can beachieved by cultivating the strains under well-defined growth conditionsin batch- and continuous cultures, and/or optimizing theexpression/activities of the individual enzymes.

Example 21 Construction of a Bacterial Vector for Expression of PAL andC4H in Lactococcus lactis

The plasmid pSH71 and derivatives thereof, which is used in thefollowing examples, is a bifunctional shuttle vector with multipleorigins of replication from Escherichia coli and Lactococcus lactis.With that, the host range specificity traverses Escherichia coli andother species of lactic acid bacteria. Though transformations inLactoccus lactis usually proceed without problems, putative difficulttransformations in other species of lactic acid bacteria can, therefore,be overcome by using Escherichia coli as an intermediate host for theconstruction of recombinant plasmids. The plasmid contains one or moremarker genes to allow the microorganism that harbour them to be selectedfrom those which do not. The selection system that is used forLactococcus lactis is based upon dominant markers, e.g. resistanceagainst erythromycin and chloramphenicol, but systems based upon genesinvolved in carbohydrate metabolism, peptidases and food grade markers,have also been described. In addition, the plasmid contains promoter-and terminator sequences that allow the expression of the recombinantgenes. Suitable promoters are taken from genes of Lactococcus lactise.g. lacA. Furthermore, the plasmid contains suitable unique restrictionsites to facilitate the cloning of DNA fragments and subsequentidentification of recombinants.

In the examples below the plasmid contains either the erythromycineresistance gene, designated as pSH71-ERY^(r), or the chloramphenicolresistance gene, designated as pSH71-CM^(r)

The gene encoding PAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-ERY^(r) vector that contains the lacA promoterfrom Lactococcus lactis. The resulting plasmid, pSH71-ERY^(r)-PAL,contains the gene encoding PAL under the control of the lacA promoterfrom Lactococcus lactis.

The gene encoding C4H, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-CM^(r) vector to yield pSH71-CM^(r)-C4H. The lacApromoter and the gene encoding C4H are reamplified as one fragment byPCR from the plasmid pSH71-CM^(r)-C4H using forward- and reverseprimers, with 5′ overhangs containing suitable restriction sites. Theintroduction of said restriction sites at the 5′ and 3′ ends of the DNAfragment allows ligation of the restricted PCR product into the digestedplasmid pSH71-ERY_(r)-PAL. The resulting plasmid, pSH71-ERY_(r)-PAL-C4H,contains the genes encoding PAL and C4H that are each under the controlof an individual lacA promoter. The sequence of the genes encoding PALand C4H is verified by sequencing of two different clones ofpSH71-ERY^(r)-PAL-C4H.

Example 22 Construction of a Bacterial Vector for Expression of TAL inLactococcus lactis

The gene encoding for TAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-TAL (example 6) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-ERY^(r) vector. The resulting plasmid,pSH71-ERY^(r)-TAL, contains the gene encoding for TAL under the controlof the lacA promoter from Lactococcus lactis. The sequence of the geneencoding for TAL is verified by sequencing of two different clones ofpSH71-ERY^(r)-TAL.

Example 23 Construction of a Bacterial Vector for Expression of 4CL andVST in Lactococcus lactis

The gene encoding 4CL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-CM^(r) vector. The resulting plasmid,pSH71-CM^(r)-4CL, contains the gene encoding for 4CL under the controlof the lacA promoter from Lactobacillus lactis.

The gene encoding VST, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pSH71-ERY^(r) vector. The resulting plasmid,pSH71-ERY^(r)-VST, contains the gene encoding VST under the control ofthe lacA promoter from Lactococcus lactis. The lacA promoter and thegene encoding VST are reamplified as one fragment by PCR from theplasmid pSH71-ERY^(r)-VST using forward- and reverse primers, with 5′overhangs containing suitable restriction sites. The introduction ofsaid restriction sites at the 5′ and 3′ ends of the DNA fragment allowsligation of the restricted PCR product into the digested plasmidpSH71-CM^(r)-4CL. The resulting plasmid, pSH71-CM^(r)-4CL-VST, containsthe genes encoding 4CL and VST that are each under the control of theirindividual lacA promoter. The sequence of the genes encoding 4CL and VSTis verified by sequencing of two different clones ofpSH71-CM^(r)-4CL-VST.

Example 24 Expression of the Pathway to Resveratrol in Lactococcuslactis

Lactococcus lactis strains are transformed with the vectors described inexamples 21, 22 and 23, separately or in combination. The transformationof the bacterial cell is conducted in accordance with methods known inthe art, for instance, by using competent cells or by electroporation(see, e.g., Sambrook et al., 1989). Transformants are selected on mediumcontaining the antibiotics erythromycin and chloramphenicol and streakpurified on the same medium.

Lactococcus lactis strain MG1363 is transformed separately with thevector pSH71-ERY^(r)-TAL (example 22), yielding the strain FSLL-TAL;with pSH71-ERY^(r)-PAL-C4H (example 21), yielding the strain FSLL-PALC4Hand with pSH71-CM^(r)-4CL-VST (example 23), yielding strain FSLL-4CLVST.In addition, Lactococcus lactis strain MG1363 is co-transformed withpSH71-ERY^(r)-TAL (example 22) and pSH71-CM^(r)-4CL-VST (example 23),and the transformed strain is named FSLL-TAL4CLVST. The same strain isalso co-transformed with pSH71-ERY^(r)-PAL-C4H (example 21), andpSH71-CM^(r)-4CL-VST (example 23), resulting in the strainFSLL-PALC4H4CLVST.

Example 25 Fermentation with Recombinant Lactococcus lactis Strains inFermentors

The recombinant yeast strains can be grown in fermenters operated asbatch, fed-batch or chemostat cultures.

Batch and Fed-Batch Cultivations

The microorganism is grown in a baffled bioreactor with a working volumeof 1.5 liters under anaerobic, aerobic or microaerobic conditions. Allcultures are incubated at 30° C., at 350 rpm. A constant pH of 6.6 ismaintained by automatic addition of 10 M KOH. Cells are grown on lactosein defined MS10 medium supplemented with the following components toallow growth under aerobic conditions: MnSO₄ (1.25×10⁻⁵ g/l), thiamine(1 mg/l), and DL-6,8-thioctic acid (2.5 mg/l). The lactose concentrationis, for example 50 g/l. The bioreactors are inoculated with cells fromprecultures grown at 30° C. in shake flasks on the medium describedabove buffered with threefold-higher concentrations of K₂HPO₄ andKH₂PO₄. Anaerobic conditions are ensured by flushing the medium with N₂(99.998% pure) prior to inoculation and by maintaining a constant flowof 50 ml/min of N₂ through the headspace of the bioreactor duringcultivation. The bioreactors used for microaerobic and aerobiccultivation are equipped with polarographic oxygen sensors that arecalibrated with air (DOT, 100%) and N₂ (DOT, 0%). Aerobic conditions areobtained by sparging the bioreactor with air at a rate of 1 vvm toensure that the DOT is more than 80%. During microaerobic experimentsthe DOT is kept constant 5% by sparging the reactor with gas composed ofa mixture of N₂ and atmospheric air, at a rate of 0.25 vvm.

Chemostat Cultures

In chemostat cultures the cells can be grown in, for example, 1-Lworking-volume Applikon laboratory fermentors at 30° C. and 350 rpm. Thedilution rate (D) can be set at different values, e.g. at 0.050 h⁻¹,0.10 h⁻¹, 0.15 h⁻¹, or 0.20 h⁻¹. The pH is kept constant, e.g at 6.6, byautomatic addition of 5 M KOH, using the growth medium described above,supplemented with antifoam (50 μl/l). The concentration of lactose canbe set at different values, e.g. is 3.0 g/l 6.0 g/l, 12.0 g/l, 15.0 g/lor 18.0 g/l. The bioreactor is inoculated to an initial biomassconcentration of 1 mg/l and the feed pump is turned on at the end of theexponential growth phase.

An anaerobic steady state is obtained by introducing 50 ml/min of N₂(99.998% pure) into the headspace of the bioreactor. Different anoxicsteady states can obtained by sparging the reactor with 250 ml/min ofgas composed of N₂ (99.998% pure) and atmospheric air at various ratios.The oxygen electrode is calibrated by sparging the bioreactor with air(100% DOT) and with N₂ (0% DOT).

For all conditions, the gas is sterile filtered before being introducedinto the bioreactor. The off gas is led through a condenser cooled tolower than −8° C. and analyzed for its volumetric content of CO₂ and O₂by means of an acoustic gas analyser.

Cultivations are considered to be in steady state after at least 5residence times, and if the concentrations of biomass and fermentationend products remain unchanged (less than 5% relative deviation) over thelast two residence times.

Example 26 Extraction and Analysis of Resveratrol in Lactococcus Lactis

Extraction and analysis is performed using the methods as described inexamples 14 and 15.

Example 27 Construction of a Fungal Vector for Expression of PAL and C4Hin Species Belonging to the Genus Aspergillus

The plasmid that is used in the following examples, is derived frompARp1 that contains the AMA1 initiating replication sequence fromAspergillus nidulans, which also sustains autonomous plasmid replicationin A. niger and A. oryzae (Gems et al., 1991). Moreover, the plasmid isa shuttle vector, containing the replication sequence of Escherichiacoli, and the inherent difficult transformations in Aspergillus nigerand Aspergillus oryzae can therefore overcome by using Escherichia colias an intermediate host for the construction of recombinant plasmids.The plasmid contains one or more marker genes to allow the microorganismthat harbour them to be selected from those which do not. The selectionsystem can be either based upon dominant markers e.g. resistance againsthygromycin B, phleomycin and bleomycin, or heterologous markers e.gamino acids and the pyrG gene. In addition the plasmid containspromoter- and terminator sequences that allow the expression of therecombinant genes. Suitable promoters are taken from genes ofAspergillus nidulans e.g. alcA, glaA, amy, niaD, and gpdA. Furthermore,the plasmid contains suitable unique restriction sites to facilitate thecloning of DNA fragments and subsequent identification of recombinants.

The plasmid used in the following examples contains the strongconstitutive gpdA-promoter and auxotropic markers, all originating fromAspergillus nidulans; the plasmid containing the gene methG that isinvolved in methionine biosynthesis, is designated as pAMA1-MET; theplasmid containing the gene hisA that is involved in histidinebiosynthesis, is designated as pAMA1-HIS.

The gene encoding PAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-MET vector that contains the gpdA promoter fromAspergillus nidulans. The resulting plasmid, pAMA1-MET-PAL contains thegene encoding PAL under the control of the gpdA promoter fromAspergillus nidulans.

The gene encoding C4H, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-PAL-C4H (example 3) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-HIS vector to yield pAMA1-HIS-C4H. The gpdApromoter and the gene encoding C4H are reamplified as one fragment byPCR from the plasmid pAMA1-HIS-C4H using forward- and reverse primers,with 5′ overhangs containing suitable restriction sites. Theintroduction of said restriction sites at the 5′ and 3′ ends of the DNAfragment allows ligation of the restricted PCR product into the digestedplasmid pAMA1-MET-PAL. The resulting plasmid, pAMA1-MET-PAL-C4H,contains the genes encoding PAL and C4H that are each under the controlof an individual pgdA promoter from Aspergillus nidulans. The sequenceof the genes encoding PAL and C4H is verified by sequencing of twodifferent clones of pAMA1-MET-PAL-C4H.

Example 28 Construction of a Fungal Vector for Expression of TAL inSpecies Belonging to the Genus Aspergillus

The gene encoding for TAL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-URA-TAL (example 6) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-MET vector. The resulting plasmid, pAMA1-MET-TAL,contains the gene encoding for TAL under the control of the gpdApromoter from Aspergillus nidulans. The sequence of the gene encodingfor TAL is verified by sequencing of two different clones ofpAMA1-MET-TAL.

Example 29 Construction of a Fungal Vector for Expression of 4CL and VSTin species belonging to the genus Aspergillus

The gene encoding 4CL, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5), usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-HIS vector that contains the gpdA promoter fromAspergillus nidulans. The resulting plasmid, pAMA1-HIS-4CL contains thegene encoding 4CL under the control of the gpdA promoter fromAspergillus nidulans.

The gene encoding VST, isolated as described in example 1, isreamplified by PCR from the plasmid pESC-TRP-4CL-VST (example 5) usingforward- and reverse primers, with 5′ overhangs containing suitablerestriction sites. The introduction of said restriction sites at the 5′and 3′ ends of the gene allows ligation of the restricted PCR productinto a digested pAMA1-MET vector to yield pAMA1-MET-VST. The gpdApromoter and the gene encoding VST are reamplified as one fragment byPCR from the plasmid pAMA1-MET-VST using forward- and reverse primers,with 5′ overhangs containing suitable restriction sites. Theintroduction of said restriction sites at the 5′ and 3′ ends of the DNAfragment allows ligation of the restricted PCR product into the digestedplasmid pAMA1-HIS-4CL. The resulting plasmid, pAMA1-HIS-4CL-VST,contains the genes encoding 4CL and VST that are each under the controlof an individual pgdA promoter from Aspergillus nidulans. The sequenceof the genes encoding 4CL and VST is verified by sequencing of twodifferent clones of pAMA1-HIS-4CL-VST.

Example 30 Expression of the Pathway to Resveratrol in Aspergillus niger

Aspergillus niger strains are transformed with the vectors described inexamples 27, 28 and 29, separately or in combination. The transformationof the fungal cell is conducted in accordance with methods known in theart, for instance, by electroporation or by conjugation (see, e.g.,Sambrook et al., 1989). Transformants are selected on minimal mediumlacking methionine and/or histidine.

A strain of Aspergilus niger that is auxotrophic for histidine andmethionine, for instance, strain FGSC A919 (see www.fgsc DOT net)(“.”replaced with “DOT” to inactivate URL), is transformed separately withthe vector pAMA1-MET-TAL (example 28), yielding the strain FSAN-TAL;with pAMA1-MET-PAL-C4H (example 27), yielding the strain FSAN-PALC4H andwith pAMA1-HIS-4CL-VST (example 29), yielding strain FSAN-4CLVST. Inaddition, Aspergillus niger strain FGSC A919 is co-transformed withpAMA1-MET-TAL (example 28) and pAMA1-HIS-4CL-VST (example 29), and thetransformed strain is named FSAN-TAL4CLVST. The same strain is alsoco-transformed with pAMA1-MET-PAL-C4H (example 27), andpAMA1-HIS-4CL-VST (example 29), resulting in the strainFSAN-PALC4H4CLVST.

Example 31 Expression of the Pathway to Resveratrol in Aspergillusoryzae

A strain of Aspergillus oryzae that contains a native set of genesencoding for PAL, C4H and 4CL (Seshime et al., 2005) and that isauxotrophic for methionine, is transformed with the vector pAMA1-MET-VST(example 29), yielding the strain FSAO-VST. The transformation of thefungal cell is conducted in accordance with methods known in the art,for instance, by electroporation or by conjugation (see, e.g., Sambrooket al., 1989). Transformants are selected on minimal medium lackingmethionine.

Example 32 Fermentation with Recombinant Strains of Aspergillus nigerand Aspergillus oryzae in Fermentors

The recombinant yeast strains can be grown in fermenters operated asbatch, fed-batch or chemostat cultures.

Batch and Fed-Batch Cultivations

The microorganism is grown in a baffled bioreactor with a working volumeof 1.5 liters under aerobic conditions. All cultures are incubated at30° C., at 500 rpm. A constant pH of 6.0 is maintained by automaticaddition of 10 M KOH, and aerobic conditions are obtained by spargingthe bioreactor with air at a rate of 1 vvm to ensure that the DOT ismore than 80%. Cells are grown on glucose in defined medium consistingof the following components to allow growth in batch cultivations: 7.3g/l (NH₄)₂SO₄, 1.5 g/l KH₂PO₄, 1.0 g/l MgSO₄.7H₂O, 1.0 g/l NaCl, 0.1 g/lCaCl₂.2H₂O, 0.1 ml/l Sigma antifoam, 7.2 mg/l ZnSO₄.7H₂O, 1.3 mg/lCuSO₄.5H₂O, 0.3 mg/l NiCl₂.6H₂O, 3.5 mg/l MnCl₂.4H₂O and 6.9 mg/lFeSO₄.7H₂O. The glucose concentration is, for example, 10-20-, 30-, 40-or 50 g/l. To allow growth in fed-batch cultivations the medium iscomposed of: 7.3 g/l (NH₄)₂SO₄, 4.0 g/l KH₂PO₄, 1.9 g/l MgSO₄.7H₂O, 1.3g/l NaCl, 0.10 g/l CaCl₂.2H₂O, 0.1 ml/l Sigma antifoam, 7.2 mg/lZnSO₄.7H₂O, 1.3 mg/l CuSO₄.5H₂O, 0.3 mg/l NiCl₂.6H₂O, 3.5 mg/lMnCl₂.4H₂O and 6.9 mg/l FeSO₄.H₂O in the batch phase. The reactor isthen fed with, for example, 285 g/kg glucose and 42 g/kg (NH₄)₂SO₄.

Free mycelium from a pre-batch is used for inoculating the batch- andfed-batch cultures. A spore concentration of 2.10⁹ spores/l is used forinoculation of the pre-batch culture at pH 2.5. Spores are obtained bypropagation of freeze-dried spores onto 29 g rice to which the followingcomponents are added: 6 ml 15 g/l sucrose, 2.3 g/l (NH₄)₂SO₄, 1.0 g/lKH₂PO₄, 0.5 g/l MgSO₄.7H₂O, 0.50 g/l NaCl, 14.3 mg/l ZnSO₄.7H₂O, 2.5mg/CuSO₄.5H₂O, 0.50 mg/l NiCl₂.6H₂O, and 13.8 mg/l FeSO₄.7H₂O. Thespores are propagated at 30° C. for 7-14 days to yield a black layer ofspores on the rice grains and are harvested by adding 100 ml of 0.1%Tween 20 in sterile water. For all conditions, the gas is sterilefiltered before being introduced into the bioreactor. The off gas is ledthrough a condenser cooled to lower than −8° C. and analyzed for itsvolumetric content of CO₂ and O₂ by means of an acoustic gas analyser.

Chemostat Cultures

In chemostat cultures the cells can be grown in, for example, 1.5-Lworking-volume Biostat B laboratory fermentors at 30° C. and 500 rpm. Aconstant pH of 6.0 is maintained by automatic addition of 10 M KOH, andaerobic conditions are obtained by sparging the bioreactor with air at arate of 1 vvm to ensure that the DOT is more than 80%. The dilution rate(D) can be set at different values, e.g. at 0.050 h⁻¹, 0.10 h⁻¹, 0.15h⁻¹, or 0.20 h⁻¹. The pH is kept constant, e.g at 6.6, by automaticaddition of 10 M KOH, using a minimal growth medium with the followingcomponents: 2.5 g/l (NH₄)₂SO₄, 0.75 g/l KH₂PO₄, 1.0 g/l MgSO₄.7H₂O, 1.0g/l NaCl, 0.1 g/l CaCl₂.2H₂O, 0.1 ml/l Sigma antifoam, 7.2 mg/lZnSO₄.7H₂O, 1.3 mg/l CuSO₄.5H₂O, 0.3 mg/l NiCl₂.6H₂O, 3.5 mg/lMnCl₂.4H₂O and 6.9 mg/l FeSO₄.7H₂O. The concentration of glucose can beset at different values, e.g. is 3.0 g/l 6.0 g/l, 12.0 g/l, 15.0 g/l or18.0 g/l. The bioreactor is inoculated with free mycelium from apre-batch culture as described above, and the feed pump is turned on atthe end of the exponential growth phase.

For all conditions, the gas is sterile filtered before being introducedinto the bioreactor. The off gas is led through a condenser cooled tolower than −8° C. and analyzed for its volumetric content of CO₂ and O₂by means of an acoustic gas analyser.

Cultivations are considered to be in steady state after at least 5residence times, and if the concentrations of biomass glucose andcomposition of the off-gas remain unchanged (less than 5% relativedeviation) over the last two residence times.

Example 33 Extraction and Analysis of Resveratrol in Aspergillus nigerand Aspergillus oryzae

Extraction and analysis is performed using the methods as described inexamples 14 and 15.

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The following is a summary of the nucleotide and amino acid sequencesappearing herein:

-   SEQ ID NO: 1 is a nucleotide sequence from Arabidopsis thaliana    encoding a phenylalanine ammonia lyase (PAL2).-   SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1.-   SEQ ID NO: 3 is a nucleotide sequence from Arabidopsis thaliana    encoding a cinnamate 4-hydroxylase (C4H).-   SEQ ID NO: 4 is the amino acid sequence encoded by SEQ ID NO: 3.-   SEQ ID NO: 5 is a nucleotide sequence from Arabidopsis thaliana    encoding a 4-coumarate:CoenzymeA ligase (4CL1).-   SEQ ID NO: 6 is the amino acid sequence encoded by SEQ ID NO: 5.-   SEQ ID NO: 7 is a nucleotide sequence from Rheum tataricum encoding    a resveratrol synthase (VST).-   SEQ ID NO: 8 is the amino acid sequence encoded by SEQ ID NO: 7.-   SEQ ID NO: 9 is a nucleotide sequence from Rheum tataricum encoding    a resveratrol synthase (VST), which is codon-optimized for    expression in S. cerevisiae.-   SEQ ID NO: 10 is the amino acid sequence encoded by SEQ ID NO: 9.-   SEQ ID NO: 11 is a nucleotide sequence from Rhodobacter capsulatus    encoding a tyrosine ammonia lyase (TAL).-   SEQ ID NO: 12 is the amino acid sequence encoded by SEQ ID NO: 11.-   SEQ ID NO: 13 is a nucleotide sequence from Rhodobacter capsulatus    encoding a tyrosine ammonia lyase (TAL), which is codon-optimized    for expression in S. cerevisiae.-   SEQ ID NO: 14 is the amino acid sequence encoded by SEQ ID NO: 13.-   SEQ ID NO: 15 is a nucleotide sequence from S. cerevisiae encoding a    NADPH:cytochrome P450 reductase (CPR1).-   SEQ ID NO: 16 is the amino acid sequence encoded by SEQ ID NO: 15.-   SEQ ID NO: 17 is a nucleotide sequence from Arabidopsis thalianus    encoding a NADPH:cytochrome P450 reductase (AR2).-   SEQ ID NO: 18 is the amino acid sequence encoded by SEQ ID NO: 17.-   SEQ ID NOs 19-32 are primer sequences appearing in Table 1, Example    1.-   SEQ ID NOs 33-34 are primer sequences appearing in Example 16.-   SEQ ID NOs 35-38 are primer sequences appearing in Example 17

The invention claimed is:
 1. An isolated or non-naturally occurringmicro-organism, wherein the micro-organism has an engineered operativemetabolic pathway producing resveratrol, or an oligomeric orglycosidically-bound derivative thereof, wherein the engineeredoperative metabolic pathway produces: a) 4-coumaric acid fromL-phenylalanine catalysed by a phenylalanine ammonia lyase and acinnamate 4-hydroxylase expressed in the micro-organism or from tyrosinecatalysed by a phenylalanine ammonia lyase or a tyrosine ammonia lyaseexpressed in said micro-organism; and b) 4-coumaroyl-CoA from 4-coumaricacid catalysed by a 4-coumarate-CoA ligase expressed in saidmicro-organism; wherein the micro-organism is cultured in a media with acarbon substrate and does not require an external source ofphenylalanine, tyrosine, cinnamic acid or coumaric acid, and resveratrolis produced from the 4-coumaroyl-CoA by a resveratrol synthase expressedin the micro-organism.
 2. The micro-organism as claimed in claim 1,wherein said resveratrol is produced in a reaction catalysed by anenzyme in which endogenous malonyl-CoA is a substrate.
 3. Themicro-organism as claimed in claim 1, wherein said resveratrol synthaseis expressed in said micro-organism from a nucleic acid coding for saidenzyme which is not native to the micro-organism.
 4. The micro-organismas claimed in claim 1, wherein said cinnamate 4-hydroxylase is expressedin said micro-organism from a nucleic acid coding for said enzyme whichis not native to the micro-organism.
 5. The micro-organism as claimed inclaim 1, wherein said phenylalanine ammonia lyase is expressed in saidmicro-organism from a nucleic acid coding for said enzyme which is notnative to the micro-organism.
 6. The micro-organism as claimed in claim1, wherein a native NADPH:cytochrome P450 reductase (CPR) isoverexpressed in said micro-organism or wherein the micro-organismcomprises a recombinant CPR.
 7. The micro-organism as claimed in claim1, wherein at least one copy of a genetic sequence encoding the tyrosineammonia lyase is operatively linked to an expression signal not nativelyassociated with said genetic sequence in said organism.
 8. Themicro-organism as claimed in claim 1, wherein at least one copy of agenetic sequence encoding the phenylalanine ammonia lyase is operativelylinked to an expression signal not natively associated with said geneticsequence in said organism.
 9. The micro-organism as claimed in claim 1,wherein at least one copy of a genetic sequence encoding the cinnamate4-hydroxylase is operatively linked to an expression signal not nativelyassociated with said genetic sequence in said organism.
 10. Themicro-organism as claimed in claim 1, wherein at least one copy of agenetic sequence encoding the 4-coumarate-CoA ligase is operativelylinked to an expression signal not natively associated with said geneticsequence in said organism.
 11. The micro-organism as claimed in claim 1,wherein at least one copy of a genetic sequence encoding the resveratrolsynthase is operatively linked to an expression signal not nativelyassociated with said genetic sequence in said organism.
 12. Themicro-organism as claimed in claim 1, containing one or more copies ofan heterologous DNA sequence encoding phenylalanine ammonia lyaseoperatively associated with an expression signal, and containing one ormore copies of an heterologous DNA sequence encodingcinnamate-4-hydroxylase operatively associated with an expressionsignal, and containing one or more copies of an heterologous DNAsequence encoding 4-coumarate CoA-ligase operatively associated with anexpression signal, and containing one or more copies of an heterologousDNA sequence encoding resveratrol synthase operatively associated withan expression signal.
 13. The micro-organism as claimed in claim 1,lacking cinnamate-4-hydroxylase activity, and containing one or morecopies of a heterologous DNA sequence encoding tyrosine ammonia lyaseoperatively associated with an expression signal, and containing one ormore copies of an heterologous DNA sequence encoding 4-coumarateCoA-ligase operatively associated with an expression signal, andcontaining one or more copies of an heterologous DNA sequence encodingresveratrol synthase operatively associated with an expression signal.14. The micro-organism of claim 1, comprising expressible nucleotidesequences encoding, and said micro-organism is capable of producing,phenylalanine or tyrosine ammonia lyase, cinnamate 4-hydroxylase,4-coumarate-CoA ligase and resveratrol synthase.
 15. The micro-organismof claim 1, wherein said 4-coumaric acid is produced from trans-cinnamicacid by cinnamate 4-hydroxylase, or said 4-coumaric acid is producedfrom tyrosine by phenylalanine ammonia lyase or tyrosine ammonia lyase,and 4-coumaroyl-CoA is formed in a reaction catalysed by an enzyme inwhich ATP and CoA are substrates and ADP is a product catalysed by4-coumarate-CoA ligase.
 16. The microorganism of claim 1 which is abacterium.
 17. The microorganism of claim 16 which is a bacteriumbelonging to a genus selected from the group consisting of Bacillus,Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Acetobacter,Acinetobacter, and Pseudomonas.
 18. The microorganism of claim 16 whichis an Escherichia coli.
 19. The microorganism of claim 1 which is ayeast belonging to a genus selected from the group consisting ofSaccharomyces, Kluyveromyces, Pichia, Debaromyces, Hansenula, Pichia,Zygosaccharomyces and Schizosaccharomyces.
 20. The microorganism ofclaim 1 which is a yeast belonging to the genus Saccharomyces.
 21. Themicroorganism of claim 1 which is a Saccharomyces cerevisiae.
 22. Themicroorganism of claim 1 which is a filamentous fungus belonging to agenus selected from the group consisting of Rhizopus, Fusidium,Gibberella, and Trichoderma.
 23. The microorganism of claim 1, whereinthe micro-organism is capable of producing 0.44-0.53 μg of resveratrolper gram dry weight.